The present invention relates to a novel glycopeptide consisting of a sugar moiety having a structural formula ##STR2## and a peptide moiety linking to the sugar moiety through N-glycoside bond and a process of isolating it. The present invention relates also to an agent of inducing glomerulonephritis in an animal and to a method of preparing an experimental model for study on human glomerulonephritis.
It is very important in medical and pharmaceutical fields, especially for an development of therapeutic treatment of human disease, screening of medicines or the like, to prepare stably, plentifully and economically an experimental model, in other words, an experimental animal having lesions which are strikingly similar to spectrum of human disease. Recently, the supply of experimental models such as, for example, rat with hypertension, mouse with diabetes, rat with digestive ulcer or the like has contributed remarkably to the development of therapeutic treatments.
Although, with respect to human glmerulonephritis, many experimental models have been reported, most of them are those about acute glomerulonephritis and have only a similarity in morphologic changes but no similarity in clinical functions such as metabolism, etc. Therefore, they cannot be utilized for the developments of diagnosis and therapeutic agents.
It is well known that there is Masugi's glomerulonephritis as one of the experimental models. Masugi's glomerulonephritis is such phenomenon that glomerulonephritis is induced accompanying with proteinuria by injecting a serum which is obtainable from steps of (1) injecting a homogenate of kidney excised from normal animal to heterologous animal in the rate of one or two times a week for about 2 months, of (2) collecting blood from the animal and of (3) separating the serum from the blood, to a normal animal homologous to the animal from which kidney is excised. As apparent from the course of inducing glomerulonephritis, it has been believed that Masugi's glomerulonephritis is induced through anitgen-antibody reaction using the homogenate of kidney.
Thereafter, many attempts have been made for purifying the homogerate to isolate a complete antigen substance that prepares "nephrotoxic antiserum" having an ability to induce glomerulonephritis. As the results of anatomical attempts, it has been found that the complete antigen substance is mainly present in glomerulus of kidney, especially glomerular basement membrane (GBM). Although many attempts from the point of chemistry have been made, the complete antigen substance has not been made clear yet.
Spiro reported that two glycopeptides can be isolated from bovine GBM [Spiro, R. G., "J. Biol. Chem.", Vol. 242, No. 8, 1923-1932 (1967)].
Spiro's procedure for isolating the two glycopeptides is as follows. Firstly, GBM is separated from kidney according to Krakower and Greenspon's method [Krakower and Greenspon "Arch. Path" 51, 629-639, (1951)]. The separated GBM is dispersed in 0.1 M Tris acetate buffer and digested with collagenase at pH of 7.4 and temperature of 37.degree. C. The supernatant liquid obtainable from centrifuging the digestion solution is conditioned at pH 7.8 and then is digested with pronase at 37.degree. C. Thus digestion solution is centrifuged to collect the supernatant liquid.
The supernatant liquid thus obtained is fractionated with Sephadex G-25. As shown in FIG. 1 of the literature mentioned above, two fractions which have sugar peaks 1 and 2 (by anthrone) respectively are obtained by this fractionation. The fraction (sugard peak 1) belonging to the void volume of Sephadex G-25 is further fractionated with Sephadex G-50. As shown in FIG. 5 of the literature mentioned above, sugar peak 1 is divided into two sugar peaks (by anthrone), one of them belonging to the void volume of Sephadex G-50.
Spiro found from this procedure two glycopeptides from the sugar peak 2 in FIG. 1 and the right sugar peak in FIG. 5. The glycopeptide of the sugar peak 2 is disaccharide comprising glucose and galactose in the ratio of 1:1 and one molecule of hydroxylysin, while the glycopeptide of the another sugar peak is heteropolysaccharide comprising galactose, mannose, glucosamine and fucose. However, both of them have no biological activity.
Spiro discarded the left sugar peak in FIG. 5 as impurities according to the common knowledge in this field because it belongs to the void volume.
The inventor has studied an establishment of an experimental model for human glomerulonephritis, especially for adult human glomerulonephritis.
It was found surprisingly that there is contained a substance having an ability to induce glomerulonephritis in animal in the left sugar peak in FIG. 5 which has been discarded by Spiro and that this substance is different quite from the two glycopeptides isolated by Spiro. It was found also that the biological activity of glycopeptide of the present invention is displayed by injecting the glycopeptide directly to an animal.
The inventor termed such biological activity of the glycopeptide of the present invention "nephrotogenicity" against that the biological activity in Masugi's glomerulonephritis was termed "nephrotoxicity".